Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.

pgen.1006446.g005: High-density aCGH results and breakpoint junction in Family 3 with apparently balanced insertion in the mother.Upper panel: High-density aCGH showed the deletion at 7p15.2p14.3 in P3, and a small deletion in Mat3 at the distal boundary of the large deletion in her child P3. Lower panel: deletion breakpoint junction sequences shared in P3 and Mat3. An 815 bp insertion from chr9:5874574–5875388 was observed at this junction. (+), sequences in the positive strand in the hg19 reference genome. Jct1, Junction1.

Mentions:
In Family 3, Proband 3 (P3) showed a ~4.588 Mb deletion at 7p15.2p14.3 from array results; this deletion was further found by FISH analysis to be inherited from Mother 3 (Mat3) with apparently balanced insertion from chr7 into 9p24 (S9A Fig, S1 Table). Upon careful interpretation of high-density aCGH results, a small deletion (~4 kb) was observed in the mother at the exact boundary of the deletion in her child (Fig 5). We were able to fine map the identical deletion breakpoint junction present in both P3 and Mat3. Interestingly, an 815 bp insertion from 9p24 (chr9:5874574–5875388) was found at the chr7 junction sequences (Jct1)–the potential insertion locus observed from FISH in Mat3 (Fig 5). We further performed high-density aCGH in both Mat3 and P3 targeting the entire short arm of chr9. No promising CNVs were identified in either Mat3 or P3, however, three probes covering chr9:5874574–5875388 showed elevated ratio only in P3 but not Mat3 (S9B Fig). Based on this observation, we suspected an exchange of genetic material between chr7 and chr9 in the mother Mat3 –the ~4.588 Mb fragment from 7p15.2p14.3 was inserted to chr9, replaced by a small fragment from chr9p24.1 (815 bp from chr9:5874574–5875388). Note that the large fragment of 7p15.2p14.3 broke and re-joined during the inserting process based on the observation of mapped breakpoint junction 2 (Jct2), and additional junctions(s) should be present that connect the inserted fragments from chr7 to chr9 except for the mapped junction 3 (Jct3, S9C Fig).

pgen.1006446.g005: High-density aCGH results and breakpoint junction in Family 3 with apparently balanced insertion in the mother.Upper panel: High-density aCGH showed the deletion at 7p15.2p14.3 in P3, and a small deletion in Mat3 at the distal boundary of the large deletion in her child P3. Lower panel: deletion breakpoint junction sequences shared in P3 and Mat3. An 815 bp insertion from chr9:5874574–5875388 was observed at this junction. (+), sequences in the positive strand in the hg19 reference genome. Jct1, Junction1.

Mentions:
In Family 3, Proband 3 (P3) showed a ~4.588 Mb deletion at 7p15.2p14.3 from array results; this deletion was further found by FISH analysis to be inherited from Mother 3 (Mat3) with apparently balanced insertion from chr7 into 9p24 (S9A Fig, S1 Table). Upon careful interpretation of high-density aCGH results, a small deletion (~4 kb) was observed in the mother at the exact boundary of the deletion in her child (Fig 5). We were able to fine map the identical deletion breakpoint junction present in both P3 and Mat3. Interestingly, an 815 bp insertion from 9p24 (chr9:5874574–5875388) was found at the chr7 junction sequences (Jct1)–the potential insertion locus observed from FISH in Mat3 (Fig 5). We further performed high-density aCGH in both Mat3 and P3 targeting the entire short arm of chr9. No promising CNVs were identified in either Mat3 or P3, however, three probes covering chr9:5874574–5875388 showed elevated ratio only in P3 but not Mat3 (S9B Fig). Based on this observation, we suspected an exchange of genetic material between chr7 and chr9 in the mother Mat3 –the ~4.588 Mb fragment from 7p15.2p14.3 was inserted to chr9, replaced by a small fragment from chr9p24.1 (815 bp from chr9:5874574–5875388). Note that the large fragment of 7p15.2p14.3 broke and re-joined during the inserting process based on the observation of mapped breakpoint junction 2 (Jct2), and additional junctions(s) should be present that connect the inserted fragments from chr7 to chr9 except for the mapped junction 3 (Jct3, S9C Fig).

Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.